Biochemical and mutational analyses of a Trametes pyranose oxidase and comparison of its mutants in breadmaking

Biochemical and mutational analyses of a Trametes pyranose oxidase and comparison of its mutants... Pyranose oxidase (POx) is a homotetrameric flavoprotein that catalyzes the oxidation of pyranose-configured sugars at position C-2 to corresponding 2-ketoaldoses. The wide substrate specificity makes POx potential for application in various biotechnological industries. In the present study we reported the gene cloning and heterologous expression of a POx from the basidiomycete Trametes sp. and functionally expressed the gene in Escherichia coli BL21(DE3). Based on sequence alignment, three residues were chosen for site-directed mutagenesis to obtain two single mutants (K312E and E539K) and two double mutants (T166A/E539K and K312E/E539K). In comparison to the wild-type, K312E shifted its optimal pH to 5.5 while the optimal temperature of E539K and K312E/E539K increased by 10 °C. The mutants retained more activities over broader pH ranges and higher temperatures and catalyzed d-glucose at higher efficiency (5800‒12,667 M−1 s−1 for the mutants versus 5083 M−1 s−1 for the wild-type). The recombinant POx and its mutants were all useful in gluten agglomeration and enlarging the loaf volume, which depends on the amounts of enzymes added. Interestingly, adding the same amount (0.5 nkat/g of flour) of wild-type and mutant enzymes differed in the change of loaf volumes, pinpointing that the catalytic activity is not the sole determinant in applying POx in breadmaking. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Biochemical and mutational analyses of a Trametes pyranose oxidase and comparison of its mutants in breadmaking

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Publisher
Springer Berlin Heidelberg
Copyright
Copyright © 2018 by The Author(s)
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
eISSN
2191-0855
D.O.I.
10.1186/s13568-018-0570-y
Publisher site
See Article on Publisher Site

Abstract

Pyranose oxidase (POx) is a homotetrameric flavoprotein that catalyzes the oxidation of pyranose-configured sugars at position C-2 to corresponding 2-ketoaldoses. The wide substrate specificity makes POx potential for application in various biotechnological industries. In the present study we reported the gene cloning and heterologous expression of a POx from the basidiomycete Trametes sp. and functionally expressed the gene in Escherichia coli BL21(DE3). Based on sequence alignment, three residues were chosen for site-directed mutagenesis to obtain two single mutants (K312E and E539K) and two double mutants (T166A/E539K and K312E/E539K). In comparison to the wild-type, K312E shifted its optimal pH to 5.5 while the optimal temperature of E539K and K312E/E539K increased by 10 °C. The mutants retained more activities over broader pH ranges and higher temperatures and catalyzed d-glucose at higher efficiency (5800‒12,667 M−1 s−1 for the mutants versus 5083 M−1 s−1 for the wild-type). The recombinant POx and its mutants were all useful in gluten agglomeration and enlarging the loaf volume, which depends on the amounts of enzymes added. Interestingly, adding the same amount (0.5 nkat/g of flour) of wild-type and mutant enzymes differed in the change of loaf volumes, pinpointing that the catalytic activity is not the sole determinant in applying POx in breadmaking.

Journal

AMB ExpressSpringer Journals

Published: Mar 13, 2018

References

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